The present invention relates to plant molecular biology and the genetic manipulation of plants, particularly to novel defense-related signaling genes and proteins and their uses in regulating cell death and disease resistance in plants.
A host of cellular processes enable plants to defend themselves from disease caused by pathogenic agents. These processes apparently form an integrated set of resistance mechanisms that is activated by initial infection and serves to limit further spread of the invading pathogenic microorganism.
Generally, after a plant recognizes a potentially pathogenic microbe the plant responds by inducing several local responses in the cells immediately surrounding the infection site. Genetic factors in both the host and the pathogen determine the specificity of these local responses, which can be very effective in limiting the spread of infection. The most commonly observed resistance response is termed the xe2x80x9chypersensitive responsexe2x80x9d (HR). In the hypersensitive response, cells contacted by the pathogen (and often neighboring cells) rapidly collapse and dry in a necrotic fleck. Other responses include the deposition of callose, the physical thickening of cell walls by lignification, and the synthesis of various small molecules and proteins with antibiotic properties. Components of many of these responses are deployed outside the plasma membrane and therefore need to be secreted.
Necrosis in plants also results from other causes. Many environmental and genetic factors can cause general leaf necrosis in maize and other plants. In addition, numerous recessive and dominant mutants have been reported which cause discrete necrotic lesions to form. These xe2x80x9clesion mutantsxe2x80x9d produce a phenotype that mimics disease lesions caused by various pathogenic organisms, even in the absence of any invading pathogens. Thus, lesion mutants are also called xe2x80x9clesion mimics,xe2x80x9d xe2x80x9cdisease mimics,xe2x80x9d or xe2x80x9cdisease lesion mimics.xe2x80x9d
Lesion mimic mutations of maize have been shown to be specified by more than forty independent loci. For example, lesi, a temperature-sensitive conditional lethal mutant in maize, mimics the appearance of Helminthosporium maydis lesions on susceptible maize. It is intriguing that more than two thirds of lesion mimic mutations display a partially dominant, gain-of-function inheritance, making this the largest class of dominant mutants in maize and suggesting the involvement of a signaling pathway in the induction of lesions in these mutations. Similar mutations have also been discovered in other plants, including Arabidopsis, barley and rice.
The pattern of lesion spread on leaves is a function of two factors: lesion initiation and individual lesion enlargement. For example, the lethal leaf spot-1(lls1) mutation of maize, which is inherited in a recessive monogenic fashion, is characterized by the formation of scattered, necrotic leaf spots (lesions) that expand continuously to engulf the entire tissue. Spots caused by lls1 show a striking resemblance to lesions incited by race 1 of Cochliobolus (Helminthosporium) carbonum on susceptible maize.
Despite availability of a large number of lesion mimic mutations in plants, the mechanistic basis and significance of this phenomenon have remained largely elusive. Similarly, an understanding of the molecular and cellular events that are responsible for plant disease resistance remains rudimentary. This is especially true of the events controlling the earliest steps of active plant defense, which include recognition of a potential pathogen and transfer of the cognitive signal throughout the cell and surrounding tissue.
Exocytosis is the final event in the secretory pathway. It requires the fusion of the secretory vesicle membrane with the plasma membrane and results in the release of vesicle contents from the cell interior to the outside. Targeting and fusion of transport vesicles requires many membrane bound and cytosolic factors, and much attention has been paid to SNAREs. The term SNARE is used to designate two distinct families of membrane anchored proteins that share structural motifs. While v-SNAREs are present on vesicle membranes, t-SNAREs are found mainly on the target membrane, plasma membrane for example. Both t- and v-SNAREs are anchored into their respective membranes by either a c-terminal hydrophobic domain or by post-translational attachment of lipids. Interaction of particular t- and v-SNAREs provides the specificity that is a hallmark of vesicle targeting (recognition) and fusion during exocytosis.
Most of the information on SNAP-25 function comes from studies of synapses in nerve terminals. Briefly, SNAP-25 is localized to the cytoplasmic side of presynaptic membranes. There it forms a ternary complex with syntaxin-1, another t-SNARE like itself, and VAMP-2 (also called synaptobrevin), the synaptic vesicle associated v-SNARE. A hexameric cytosolic ATPase [from the family of AAA-type ATPases, and also known as NSF (N-ethylmaleimide sensitive factor)], dissociates this complex during priming of the exocytotic complex with the assistance of another protein, SNAP (soluble NSF attachment protein). Subsequent reassembly is promoted by SNAP-25 and may drive calcium-triggered vesicle membrane fusion. See, for example, Kwong, et al. (2000) Journal of Cell Science 113:2273-2284.
In addition to phytoalexins, a number of other compounds, both proteinaceous and non-proteinaceous, are secreted in the extracellular environment in the vicinity of attempted infection. These include a number of PR proteins, precursors of callose, suberin and lignification, and a variety of phenylpropanoids and their derivatives. Impairment in the secretion of these compounds is likely to weaken plant""s resistance to pathogens. Conversely, accelerated or enhanced secretion of these molecules may lead to a more vigorous and effective resistance response. Thus, there is an art recognized need for compositions and methods that modulate the exocytosis of defense proteins or metabolites that protect plants against pathogen attack and disease.
Clearly, exocytosis is an essential cellular process with roles in many physiological functions. In addition, defective exocytosis has been implicated in diverse human diseases, including polycistic kidney disease, creutzfeld-jakob disease, cancer, tetanus and botulism. Thus compositions and methods of modulating exocytosis may contribute methods of modulating human diseases and physiology.
Generally, it is the object of the present invention to provide nucleic acids and proteins relating to plant defense-related signaling, for example, nucleic acids having a nucleotide sequence as set forth in SEQ ID NO:1, 2, 4, or 6, and polypeptides having an amino acid sequence as set forth in SEQ ID NO:3, 5, or 7. It is an object of the present invention to provide transgenic plants comprising the nucleic acids of the present invention. It is another object of the present invention to provide methods for modulating, in a transgenic plant, the expression of the nucleic acids of the present invention.
Therefore, in one aspect, the present invention relates to an isolated nucleic acid comprising a member selected from the group consisting of (a) a polynucleotide encoding a polypeptide of the present invention; (b) a polynucleotide amplified from a Zea mays nucleic acid library using the primers of the present invention; (c) a polynucleotide comprising at least 20 contiguous bases of the polynucleotides of the present invention; (d) a polynucleotide encoding a plant SNAP25 protein; (e) a polynucleotide having at least 50% sequence identity to the polynucleotides of the present invention; (f) a polynucleotide comprising at least 25 nucleotide in length which hybridizes under low stringency conditions to the polynucleotides of the present invention; and (g) a polynucleotide complementary to a polynucleotide of (a) through (f). The isolated nucleic acid can be DNA, RNA, or a synthetic analog thereof.
In another aspect, the present invention relates to vectors comprising the polynucleotides of the present invention. Also the present invention relates to recombinant expression cassettes comprising a nucleic acid of the present invention operably linked to a promoter.
In another aspect, the present invention is directed to a host cell into which has been introduced the recombinant expression cassette. In yet another aspect, the present invention relates to a transgenic plant or plant cell comprising a recombinant expression cassette with a promoter operably linked to any of the isolated nucleic acids of the present invention. For example, plants containing the recombinant expression cassette of the present invention could include but are not limited to maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice barley, and millet. The present invention also provides transgenic seed from the transgenic plant.
In another aspect, the present invention relates to an isolated protein selected from the group consisting of (a) a polypeptide comprising at least 25 contiguous amino acids of SEQ ID NO:3, 5, or 7; (b) a polypeptide which is a plant SNAP25; (c) a polypeptide comprising at least 55% sequence identity to SEQ ID NO:3, 5, or 7; (d) a polypeptide encoded by a nucleic acid of the present invention; (e) a polypeptide characterized by SEQ ID NO:3, 5, or 7; and (f) a conservatively modified variant of SEQ ID NO:3, 5, or 7.
Methods of modulating the level of protein in a plant are also provided. These methods comprise introducing into a plant cell a recombinant expression cassette comprising a polynucleotide of the present invention operably linked to a promoter; culturing the plant cell under appropriate growing conditions to produce a regenerated plant; and inducing expression of the polynucleotide for a time sufficient to modulate the protein of the present invention in the plant. For example, protein levels could be modulated in plants such as maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, and millet. The level of protein in the plant can either be increased or decreased.
The compositions of the invention include isolated nucleic acid molecules and proteins. The nucleic acid molecules of the invention are polynucleotides having the sequences set forth in SEQ ID NO:1, 2, 4, or 6, and fragments thereof. The maize Rm1 nucleotide sequence is set forth in SEQ ID NO:1. The maize SNAP25 nucleotide sequence is set forth in SEQ ID NO:2, and the maize SNAP25 amino acid sequence is set forth in SEQ ID NO:3. The rice SNAP25 nucleotide sequence is set forth in SEQ ID NO:4, and the rice SNAP25 amino acid sequence is set forth in SEQ ID NO:5. The wheat SNAP25 sequence is set forth in SEQ ID NO:6, and the wheat SNAP25 amino acid sequence is set forth in SEQ ID NO:7. The compositions of the invention also comprise fragments and variants of the disclosed sequences. A nucleotide sequence of the invention may be 26 contiguous nucleotides of a disclosed sequence, or a nucleotide sequence of the invention will have at least 75% sequence identity to a disclosed nucleotide sequence and encode a polypeptide having defense-related activity.
The nucleotide sequences of the invention may be presented in vectors or expression cassettes. In an expression cassette, the nucleotide sequence of the invention is operably linked to a heterologous promoter that drives expression in the host cell. The host cell may be a plant cell, microorganism, or animal cell.
The invention also comprises plants and plant cells containing the isolated nucleic acid molecules of the invention. An embodiment of the invention is a plant having stably incorporated into its genome at least one polynucleotide operably linked to a heterologous promoter active in the plant.